Expression of foreign genes in heterologous hosts such as plant cells has been difficult, and often detectable expression of the product is not obtained (Fischhoff et al. (1987) Bio/Technology 5:807-813; Vaeck et al. (1987) Nature 328:33-37). Explanations for poor expression include the fortuitous presence of processing sites, leading to non-functional mRNA transcripts in the transgenic host cells; differing codon usage in plants as compared to the original host; and differences in GC content in the gene sequence and resulting primary RNA transcript. DNA structure is comprised of pairs of bases, along a double helix. It is well known that total DNA from different organisms has different distributions of the four nucleotides, adenine (A), thymidine (T), guanosine (G), and cytidine (C). Within a DNA helix, adenine and thymidine form a ‘base pair’ by hydrogen bonding (an “AT base pair”). Similarly, guanosine forms base pairs only with cytidine (a “GC base pair”). The ratio of such AT base pairs to GC base pairs is often referred to as ‘GC content’, and is a percentage of the total base pairs that are comprised of GC base pairs, as opposed to AT base pairs. Similarly, ‘AT content’ refers to the percentage of base pairs that are comprised of AT base pairs. GC content varies not only between different organisms, but can vary within different regions of the DNA of a genome, and even within the different regions of a gene, such as a plant gene. Plant introns have about 70% AT (30% GC) content, whereas exons have about 50% AT (50% GC) content (Wiebauer et al. (1988) Mol. Cell Biol. 8:2042-2051).
Due to the difficulty of expressing insecticidal proteins from Bacillus thuringiensis (Bt) in plants, various synthetic genes capable of higher levels of expression in transgenic plants have been designed. Adang et al. (U.S. Pat. Nos. 5,380,831, 6,015,891) describe designing synthetic Bt insecticidal proteins to have enhanced expression in plants by modifying the gene to contain codons preferred by highly expressed plant genes. The codons used are selected with regard to the distribution frequency of codon usage employed in highly expressed plant genes. Adang et al. (U.S. Pat. No. 5,567,600) further describe similar methods where the modification comprises reducing the number of codons having GC in codon positions II and III in a region between plant polyadenylation signals. Alternatively, modifications could result in fewer occurrences of the sequence AATGAA (SEQ ID NO: 1).
Fischhoff et al. (U.S. Pat. No. 5,500,365) disclose modified Bt genes that have plant polyadenylation sequences and ATTTA (SEQ ID NO:2) sequences removed. Koziel et al. (U.S. Pat. No. 6,320,100) disclose synthetic Bt insecticidal proteins selected for optimized expression in a plant, produced using codons that most frequently encode each amino acid in maize, and having 60% GC content.